We are studying the cooperative interactions between cellular and retrovirus-encoded proteins that together regulate RNA synthesis, pre- mRNA processing, and mRNA transport; processes that are essential for virus replication and pathogenesis. Our focus is primarily directed towards identifying the gene products and mechanisms essential for human T-cell leukemia virus type I (HTLV-I) infectivity and oncogenic transformation. We have developed an infectious molecular clone of HTLV-I and have established cell culture systems to monitor virus infectivity and replication. Furthermore, primary human T-cell lines have been immortalized with cloned proviruses in order to examine how cellular signaling networks are reprogrammed by virus gene products. For example, we observed that HTLV-I-transformed T-cells show a consistent alteration in the expression pattern of the src-related kinases Lyn and Lck; the former increases and the latter decreases after transformation. Lyn, which is not normally expressed in T-cells, was found to be associated with IL2 receptor subunits and Jak3 protein in HTLV-I-transformed cells. In order to better understand early events in T-cell transformation, we are investigating HTLV-I Tax-mediated activation of cellular genes controlled by serum response factor (SRF). We found that transcriptional activation mediated by SRF requires the participation of the cellular cofactors Sap1a and CBP. The mechanisms and gene products involved in HTLV-I replication are poorly characterized due to the poor infectivity and inefficient replication of the virus in vitro. We observed that inefficient replication of HTLV-I is probably related to its poor infectivity and to the frequent generation of defective proviral genomes generated by aberrant strand transfer reactions. We are currently identifying the underlying mechanisms for these phenomena by studying the replication of provirus mutants and by characterizing recombinant viral proteins in vitro.We are also comparing the functions and molecular mechanisms of the Rev proteins encoded by various lentiviruses, including human immunodeficiency virus (HIV-1) and its relatives.This research is focused on a comparative analysis of the components and pathways that regulate retroviral mRNA splicing and transport. These processes are mediated by interactions among the viral Rev proteins, cellular factors, and cis-acting RNA elements. A comparison of HIV-1 and equine infectious anemia virus (EIAV) Rev proteins revealed that the latter mediates alternative splicing and nuclear export of viral pre-mRNA. The distantly related Rev proteins share many features in common but also display distinct differences in the ways in which they interact with RNA. We have characterized the primary RNA element bound by EIAV Rev and showed that it is quite different compared to that of other retroviruses. We have also exploited the EIAV system to identify cellular splicing factors that participate with Rev in its post- transcriptional regulation of virus gene expression. (Project transferred to BRL)AIDS TITLE: Structure and Function of Lentivirus Tat and Rev Proteins. - AIDS, gene splicing, Gene Transcription, HIV, Leukemia, retroviruses, Signal Transduction, T-lymphocytes, Virus-Cell Interactions, T-cell transformation, gene splicing, HTLV-1, mRNA Export, Protein-Tyrosine Kinase, - Human Tissues, Fluids, Cells, etc.